1. Field of the Invention
This invention relates to an apparatus and method for preparing experiments using proteins that are contained within gels. More particularly, the apparatus and method relates to dispensing minute quantities of protein-containing gels into containers, e.g., assay trays, by means of automated equipment.
2. Discussion of the Art
Membrane proteins, which reside in highly insulating lipid bilayers, catalyze vital reactions, such as solute transport, charge separation, and conversion of energy, as well as signal transduction. In so doing, membrane proteins enable rapid communication between the interior and exterior of living cells. Examples of membrane proteins include ion channels, signaling receptors, hormone receptors, light receptors, and adhesion proteins. Such membrane proteins are the targets of drugs under development to treat numerous ailments. Understanding such processes at a molecular level requires knowledge of the structures of these hydrophobic proteins at high resolution. Structures of proteins are often determined by a study of their crystals, e.g., by x-ray crystallography. Membrane proteins are difficult to crystallize, on account of their hydrophobic and/or lipophilic nature, which makes them difficult to purify in large quantity and reduces their solubility in aqueous solutions. Membrane proteins tend to be unstable at concentrations in aqueous solutions that are required for the nucleation of crystal growth by crystallization methods used for soluble proteins, i.e., those proteins that are not bound to a membrane.
Landau, E. M. and Rosenbusch, J. P. (1996) Proc. Natl Acad. Sci. USA 93, 14532-14535), describe the use of lipidic cubic phases for the crystallization of membrane proteins. According to this method, detergent solubilized membrane protein is mixed with monoolein or monopalmitolein and water or buffered solutions, followed by several centrifugation steps. This method allowed for gentle mixing of the materials over a period of two to three hours to create a viscous, bicontinuous cubic phase, a cured bilipid layer, extending in three dimensions and permeated by aqueous channels. The membrane proteins can partition into the lipid bilayer and can diffuse in three dimensions, thereby allowing them to explore many potential spatial packing configurations that can lead to crystal growth of the protein within lipidic mesophases, e.g., the lipidic cubic phase.
Rummel, G., Hardmeyer, A., Widmer, C., Chiu, M., Nollert, P., Locher, K., Pedruzzi, I., Landau, E. M., & Rosenbusch, J. P. (1998) J. Struct. Biol. 121, 82-91, describe a method for preparing cubic phases by mixing dry lipids with aqueous solutions that contain protein and detergent. The water/lipid ratios are chosen according to the known phase diagrams of the pure/lipid water systems. Cubic phase formation is achieved by centrifugation, and is considered complete once the matrix forms a transparent, nonbirefringent solid material.
Cheng, A., Hummel, B., Qiu, H. & Caffrey, M. (1998) Chem. Phys. Lipids 95, 11-21, describe a simple device for rapid and convenient hydration and mixing of small volumes (10-500 μl) of viscous hydrated lipid samples for use in X-ray diffraction/scattering and other applications. The device is a low-dead volume (3.6-11.2 μl) device that was built to facilitate maximal transfer of homogeneously hydrated lipid from the mixer into 1 mm diameter X-ray capillaries with minimal loss of water during transfer and sample manipulation. The device consists of two microsyringes joined by a small-bore coupling needle. The paper also describes a technique for determining the water content of the small volume, hydrated samples prepared with the mixer and an accessory for heating and/or degassing samples during mixing. The mixing is achieved by extruding the lipid through a small orifice. In this method, the likelihood of syringe breakage, upon which all protein is lost, is high.
In an alternative method, the lipid can be melted, mixed with a protein-containing solution by means of a spatula, and undergo centrifugation by means of a tabletop centrifuge for a period of 10 to 15 minutes. The mixing and centrifugation can be repeated several times, typically three times. The sample can then be introduced to a syringe and then dispensed by hand or with a ratcheted dispenser into capillary tubes.
The primary problems associated with dispensing protein-containing lipidic cubic phase from a syringe by hand are the inconsistency of application of the protein-containing phase and the significant manual labor required to prepare a large number of samples. Likewise, the application of precipitating agent is laborious. Furthermore, the capillary tubes had diameters of unreliable size, which decreased reproducibility of experiments. Also, only batch crystallization, in which a small quantity of protein-containing phase is immersed in a precipitating solution, was possible with capillary tubes.
WO 02/05962 A1 describes a method of transferring viscous material, such as lipidic cubic phase material from a first syringe barrel to a second syringe barrel. The transfer can be carried out by providing a first syringe barrel containing a volume of viscous material, the first syringe barrel having a first volume size; providing a coupling device; coupling the first syringe barrel with the coupling device; providing yet another syringe barrel having a different volume size from that of the first syringe barrel; coupling this second syringe barrel with the coupling device; and utilizing air pressure to transfer at least a portion of the viscous material to the second syringe barrel from the first syringe barrel.
U.S. Pat. No. 5,454,268 A describes a device that measures and dispenses accurate and reproducible volumes of normal, viscous, or low-density organic liquid. The device consists of a rigid cylindrical tube containing a main coil spring, which makes the core shaft move, a double-plunger attached on the low end of the core shaft, and a syringe barrel attached securely on the low end of the cylindrical tube. The coil spring is supported by the lower end plug through which the core shaft moves.
It would be desirable to eliminate problems such as imprecise dispensing of lipidic cubic phase in crystallization trays. It would be desirable to reduce the high level of manual labor that is necessary to prepare large numbers of crystallization experiments involving membrane bound proteins. Furthermore, it would be desirable to have an alternate method of dispensing soluble proteins or membrane proteins in other silica gels, agarose gels, sol/gels, and hydrogels that are commonly used in crystallization processes.